Project Summary: Functional mapping of spontaneous brain activity with resting-state functional connectivity (FC) analysis of fMRI data has recently become a dominant approach to mapping human brain function and continues to gain momentum. However fMRI is based on cerebral hemodynamics that is relatively indirectly coupled to neuronal activity and much slower (~0.3 Hz). Further the physiological underpinnings of FC are relatively un-resolved, such that the mechanisms and implications altered FC are often unclear. For example in ischemic stroke, it is well known that the penumbra surrounding the ischemic core has altered neurovascular coupling (NVC), complicating the interpretation of the FC deficits. As FC measures are extended further into studies of brain development, aging and disease, the importance of understanding the fundamental basis for FC will grow. We recently developed hemodynamic mapping of functional connectivity in mice using optical intrinsic signal imaging (fcOIS), and found fcOIS sensitive to several neurological diseases, including mouse models of stroke and Alzheimer's disease. However, with the advent of genetic engineering techniques for mice, there are new opportunities for extending optical wide-field imaging to calcium activity, which is >10x faster and more directly coupled to neural activity than hemoglobin. By combining calcium and hemodynamic imaging, there is the potential to quantify the relationship between cell-specific calcium dynamics and hemodynamics throughout brain regions. Further concurrent calcium and hemoglobin imaging could help resolve questions about the impact of altered NVC in diseases such as stroke, and in early brain development. However, as yet, no imaging system has been developed to examine these rich relationships throughout the mouse cortex. In this project, we will develop optical imaging hardware and software for characterizing calcium dynamics in mice engineered for genetically encoded calcium indicators (GECI's). For exemplar applications where the functional networks are changing quickly, we will quantify FC during stroke recovery and brain development, tracking the progression of both functional connectivity and neurovascular coupling (NVC). Aim 1 will develop fluorescence molecular tomography (FMT) and diffuse optical tomography (DOT) instrumentation for concurrent mapping of calcium and hemoglobin in mice. Aim 2 will optimize system performance for high speed FMT/DOT of mouse brain function. Aim 3 will establish FMT/DOT for mapping the functional networks of cell-specific calcium signals in mice with GECIs. Concurrent imaging with hemoglobin will enable mapping of neurovascular coupling. In aim 4, with establish feasibility of FMT/DOT in both stroke recovery and brain developmental. In both applications we will quantify calcium- FC and the influence of altered NVC on hemoglobin-FC.